helo

1. - Dr. Raj Khirasaria
Isolated Tissue Preparations

2. Types
In vitro Ex vivo In vivo
In situ In silico

3. Advantages
Several preparations from single animal
Small amount of drug
Drug effect is tested directly

4. Guinea pigs Rabbits
Rats & Mice Cats & Dogs
Sources

5. Isolated strips of intestine
Abundant material , more resistant &
easy to set up
Variable spontaneous activity of
different sections
Different type of pharmacological
actions can be studied

6. Muscle tone
Biophase
Contact period of the Drug with the tissue
Rate of action of a Drug

7. Process
• Complete adjustment of organ bath
• Adjustment of lever
• Animal and tissue selection
• Surgical process and collection of a tissue
• Tissue attachment to organ bath
• Relaxation time to tissue

8. • Prepare the standard drug
• Test any concentration of drug for response
• Standardize tissue response with same
dose
• Prepare DRC for standard drug
• Prepare DRC with test drug

9. Important Points to Remember
• Check Organ bath and PSS
• Writing Lever
• Dissection
• Rest
• Empty organ bath regularly
• Add drug slowly

10. • Allow drug to act
• Wash
• Put a mark on drum
• Use stopwatch during experiment
• Start with a very low concentration

11. Physiological Salt Solution
• Also called as PSS / Ringer solution
• Maintains tissue outside the animal body
• Select PSS in which tissue last longer
• Prepare the solution with the help of distilled
or double distilled or deionised water
• Prepare fresh solution

12. SOLUTION USE
Tyrode solution Mammalian smooth
muscle
Frog-Ringer solution Amphibian tissue
Ringer-Locke solution Heart muscle preparation
Krebs solution Any tissue

13. • McEwen solution - sucrose in addition to
glucose
• Krebs solution - used for any tissue
• DeJalon , Frog-Ringer and Ringer-Locke do not
contain magnesiun and phosphate
• Krebs, DeJalon and McEwen – aerated with
carbogen and used for the mammalian
isolated organ and avian skeletal muscle

14. Fast Contracting Smooth Muscle
Preparation
TISSUE DRUG
Guinea pig ileum Histamine
Rat colon Acetylcholine (ACh)
Rat uterus ACh
Rat anococcygeus ACh
Rat vas deferens ACh
Guinea pig atria Adrenaline (Adr)

15. Guinea Pig Ileum
• AIM :- To determine unknown concentration
of histamine by using guinea pig ileum
• Guinea pig ileum is most sensitive to
histamine .
• It has the spontaneous activity and specificity
is improved by using atropine (for histamine
assay) or mepyramine (for acetylcholine) in
Tyrode.

16. • Ileum is preferred because of less mesentry
attached to it, and nearly all receptors are
present.
• 10 cm of ileum attached to the cecum should
be excluded.
• Spontaneous activity of the tissue is reduced
by performing the experiment 5-7°C lower
than body temperature.

17. Rat Colon
• AIM:- To determine unknown concentration of
ACh using rat ascending/descending colon.
• Sensitivity may increase by keeping colon at
4°C for 24 hr.
• Expression of calcium-sensing receptors(CaSR)
mediates increase in inositol 1,4,5-
trisphosphate.

18. • Easy to isolate and more handling resistant.
• For ACh , most sensitive tissue is dorsal leech
muscle and frog rectus abdominis.
• Initial few centimeters of colon usually used
for bioassay of Adr and NA.

19. Guinea Pig Atria
• AIM:- To determine unknown concentration of
Adr using guinea pig atria
• Beta 2 receptors widely present in atria
• Required less trimming or slicing of the tissue
• Advantages :- Tissue thickness , easy
separation of right and left atria , good
contractile tension , stability etc

20. Rat Uterus
• AIM :-To determine unknown concentration of
ACh using rat uterus
• Induce the estrus cycle in female rat
• Treat with stilbestrol 0.1mg/kg, SC ,
24 hrs before the experiment
• Other drugs like adrenaline , histamine,
oxytocin also sensitive to uterus

21. • Response depends on the animal age due to
variation in estrus cycle .
• Other drugs like 5-HT,NA can be assayed .

22. Rat Vas Deferens
• AIM :- To determine unknown concentration
of ACh using rat vas deferens
• Described by the Henderson et al(1972) and
Hart et al(1979)
• Rat and guinea pig are suitable animals
• Animal should be fed with oats for 3 days .

23. • Adrenaline and phenylephrine contract the
vas deferens whose action is blocked by the
phentolamine .

24. Rat Anococcygeus
• AIM: To determine unknown concentration of
ACh using rat anococcygeus muscle
preparation
• Decribed by Gillespie (1972)
• It arises from sacral vertebrae and reaches to
terminal colon (near anus) .
• It has tendinous origin and do not appear soft.

25. • It has a dense adrenergic excitatory and
inhibitory innervations .
• Insenistive to histamine .

26. Slow Contracting Muscle
TISSUE DRUG
Rat fundus Seretonin (5-HT)
Frog rectus abdominis ACh
Guinea pig trachea ACh
Rat phrenic nerve diaphragm ACh
Chick biventral cervicis Neuromuscular blocking agents

27. Rat Fundus
• AIM :-To determine unknown concentration of
5-HT using rat stomach(fundus)
• Stomach fundus is most sensitive tissue
among the whole parts of stomach.
• fundus identification – gray color , above the
pink thick pylorus.
• Insensitive to histamine

28. • Animal should be fasted properly.
• Fundus contains the swallowed air.
• Sometimes it needs a stretching weight.
• Both longitudinal and circular muscles may be
used in the experiment which depends on the
transverse cut made to prepare the tissue.

29. Frog Rectus Abdominis
• AIM :- To determine unknown concentration
of ACh using frog rectus abdominis muscle.
• It’s a striated skeletal muscle sensitive to Ach
and the easiest isolated tissue to handle.
• Contains more of multiple-innervated fibers so
show the slow contraction.
• Frog, being an amphibian animal, it responds
under room temperature.

30. Guinea Pig Trachea
• AIM :- to determine unknown concentration
of ACh using guinea pig trachea
• Extraction of at least 6 cm of trachea required
• Tracheal ring is in “D” form and smooth
muscle is present in the straight line of “D”
shape.
• Response is slow to develop but last for longer
period

32. • Mainly to demonstrate the respiratory
dominant adrenoreceptor so utilized to study
the bronchodilators like Adr , theophylline.
• Ideal model to study the contractile drugs like
Ach,5-HT and histamine, and their antagonism
can be studied by drugs like Adr ,
aminophylline.

33. Rat Phrenic Nerve Diaphragm
• AIM :- To determine unknown concentration
of ACh using rat phrenic nerve diaphragm
• This is primary motor nerve of the diaphragm
which arises mainly from the 4th cervical
nerve.
• Useful for evaluation for neuromuscular
function

34. Chick Biventer-Cervicis
• AIM : - To determine neuromuscular blocking
drugs using innervated biventer-cervicis
preparation
• Biventer-cervicis – an anatomically complex
tendinous muscle
• Responds differently twitch / contraction to
different stimuli

35. Cardiac Muscle Preparation
Effect of various drugs on Isolated heart
(Langendorff’s preparation)
Effect of different drugs on Normal and
Hypodynamic Rabbit heart
Effect of inotropic and chronotropic effects of
various drugs on Frogs heart

36. Langendorff’s Preparation
• AIM :- To observe the effect of various drugs
on Isolated heart
• In 1897 , Oscar Langendorff established the
isolated mammalian heart preparation.
• Based on principle of retrograde flow in aorta
either at constant flow or constant pressure .

37. • Animals : -
-Albino Rats
-New Zealand Rabbits
-Guinea pig
• 23° ± 2° C temperature , 12:12 hr light and
dark cycle ,free access to tap water and food
ad libitum

38. • Animal is pretreated with the heparin .
• The recording is done by attaching the thread
to a strain gauge transducer , an attachment
with the student physiograph

41. Rabbit Heart
• AIM : To determine effect of different drugs
on Normal and Hypodynamic Rabbit heart
• Hypodynamic heart – defined as the heart
exhibiting subnormal power or force than the
normal one.
• Experimentally ,it is developed by the supply
of the 1/4th of Calcium chloride(CaCl2) than
the required one which reduces the heart
rate.

42. • For normal heart – McEwens solution
• For hypodynamic heart – 1/4th of CaCl2 in
McEwens solution

44. Frog Heart
• AIM :- To demonstrate the Effect of inotropic
and chronotropic effects of various drugs on
frog heart
• The heart is the most common site for the
drug target.
• Positive inotropic & chronotropic drugs like
Adr and NA .
• Negative inotropic & chronotropic drugs like
ACh .

45. Tissues/muscle Identification point Drug sensitivity Receptors
Tracheal chain Above esophagus NA ,A & Ach β2 & M2/M3
Stomach fundus Upper grey part
attached to thick red
Pylorus
5-HT > Ach >
Histamine >
Bradykinin
5HT-D
Ileum Cecum at middle part
& large intestine at
distal part
Histamine >Ach H1, Muscarinic
Ascending colon 4-7 cm from
ileocecum junction
NA>A
Ach,5-HT
β3 , 5HT-2A , 5HT4
Descending
colon
5-7cm above rectum Ach Muscarinic
Anococcygeus Thin muscle strip
arises from the sacral
vertebrae
NA, Ach, 5-HT , isoP,
No histamine
Adrenergic supply,
NANC
Vas deferens Attached to
epididymis
NA & Ach α1, Muscarinic
Uterus Clear two horn above
rectum connected to
ovary
Oxytocin,5-HT,A,NA
Ach
β , α
M2/M3

46. Rabbit Perfused Ear Artery
• Lop-eared rabbit is used
• Central artery is identified , cannulated ,
dissected & perfused .
• Used for bioassay of catecholamines

48. Rabbit Aortic Strips
• Descending thoracic aorta is used .
• Aorta is cut in a close spiral .
• 2 to 4 cms of strips are used for experiment .
• Has a very slight spontaneous tone .
• Shows no rhythmic contraction .
• Responds to Adr and NA.

49. Tissue / Organ Response Receptor
Rat isolated vas deferens and
anococcygeus
Rabbit and guinea pig isolated aorta
Rabbit isolated jejunum
Guinea pig isolated ileum
Contraction
Contraction
Inhibition
Inhibition
Postsynaptic α₁
Rabbit isolated ear artery and jejunum Inhibition Presynaptic α₂
Rabbit isolated perfused heart
Rabbit isolated jejunum
Contraction
Inhibition
β₁
Guinea pig isolated trachea
Rat isolated uterus
Inhibition
Inhibition
β₂
Mouse isolated spleen contraction
Relaxation
Both